1. Field of the Invention
The invention relates to methods for measuring the effect of growth interacting substances on microbial growth.
2. Description of the Prior Art
One well known test is the spot diffusion test in which a solution of a growth interacting substance is applied to a culture medium which has been inoculated with microbes. The resultant plate is incubated for a period of time sufficient to produce visible microbial colonies for areas of the plate where the potency of the growth interacting substance was not high enough to inhibit growth. The spot test has known deficiencies. First, it does not provide direct quantitive identification of the potency of the growth interacting substance at points of interest on the incubated plate such as the point of inhibition. Second it is limited by the degree of diffusibility of growth interacting substances into the microbial culture medium.
A second well known testing method of growth interacting substances involves the making of a series of solutions of increasing dilution of the growth interacting substance. Each solution is applied to a microbial culturing medium which has been inoculated with a microbe solution and incubated for a time sufficient to detect the effect of the solution on microbial growth. This method also suffers from known drawbacks. In the first place, it requires time consuming manual dilutions and requires the use of a large number of culture media containers. It also does not readily yield precise quantitative data on the potency of the growth interacting substance being studied. In the case of its use to detect the point of inhibition, it usually reveals only a range of potency between which growth inhibition occurs.
The spot and the dilution tests both suffer from the drawback of not readily facilitating testing of growth interacting substances to determine their effects in potencies which are less than those which completely inhibit microbe growth.
The dilution method is also used to determine the effect of growth interacting substances at less than inhibiting potencies by counting the number of surviving microbial colonies. The disadvantage of this procedure is that it requires separate tests since exposure times for survivor measurements are generally different than exposure times for inhibition measurement.
All of the above methods suffer from being time consuming because of the need for manual manipulation and because they do not readily yield continuous quantitive information on the potency of a growth interacting substance for diverse points of interest on a growth curve of the microbes being studied.
Spiral Systems, Inc. manufactures an instrument under the trademark SPIRAL PLATER for plating onto a microbial culture medium a solution containing an unknown concentration of microbes to determine the concentration of microbes in the solution by counting the resultant colonies after an incubation period. The solution is deposited on a circular culture medium plate in an Archimedes spiral. The solution decreases in volume per unit length of the spiral as the radius of the spiral increases. The apparatus is described in U.S. Pat. Nos. 3,799,844, 3,892,632, and 3,962,040. These patents are incorporated herein by reference.
A laser bacteria colony counter (Model 500A) is manufactured by Exotech Incorporated, of Gaithersburg, Md., specifically for counting the colonies which result from the plating of a microbe containing solution with the SPIRAL PLATER. The laser counter is the preferred microbe colony scanning mechanism which is used with the invention. The Model 500A is described in a brochure entitled "User Manual Model 500A Laser Bacteria Colony Counter, October 1981" by Spiral System Instruments, Inc. of Bethesda, Md., 20814. The description of the Model 500A is incorporated herein by reference.
The Food and Drug Administration pursuant to 21 CFR 036.105 requires that each manufacturer of antibiotics must run tests of each batch of newly manufactured antibiotic to determine compliance with potency standards. Implementation of this requirement entails the measurement of zones of inhibition of the test substance relative to the size of inhibition zones produced by known potencies of control substances. Measurement of these zones of inhibition is done either manually or by vidicon scanners. These tests are costly in their need for extensive time, materials, and equipment.